The goal of this proposal is to establish a foundation for understanding the roles of poly(U) polymerases (PUPs) and RNA uridylation. PUPs are conserved from fission yeast to humans and add uridines to the 3' end of RNA substrates to form U tails1-10. PUPs act on an exceptionally broad range of substrates, from microRNAs (miRNAs) to mRNAs, and the effects of U tails are astoundingly diverse. U tails can cause degradation9,14-17, stabilization18,19, altered function6, altered processing4-6,20, altered translation15,21,22, and mRNA editing10. Investigation of the roles of PUPs impacts human health. PUPs and several of their RNA substrates have been directly linked to tumorigenesis11, proliferation5,8,12, stem cell maintenance4, and immune response6,13. I will use C. elegans as model to begin to understand the biological roles of PUPs and of U tails. The experiments in this proposal will identify C. elegans PUPs and then develop both biochemical tools to identify RNA substrates and genetic tools to assess PUP biological functions. Aim 1: Identify C. elegans PUPs and begin to elucidate their biochemical and biological activities. PUPs belong to the family of beta-nucleotidyl transferases that act on RNA (rNTRs), which includes noncanonical poly(A) polymerases (PAPs)36. PUPs cannot be distinguished from noncanonical PAPs by sequence similarity alone. The C. elegans genome encodes six additional noncanonical rNTRs, as determined by sequence similarity to known PUPs and noncanonical PAPs. Experiments in Aim 1 will identify C. elegans PUPs and then further examine PUP activity in vitro. Genetic studies in Aim 1 will begin to address the functions of PUP-2, PUP-3, and additional germline PUPs by using diagnostic phenotypes to assess the functional crosstalk between PUPs in one tissue. Aim 2: Identify RNA substrates of PUP-2 and of PUP-3 on a genome-wide scale. PUPs can act on diverse RNAs and elicit a variety of effects. Uridylation triggers degradation of mRNAs and siRNAs14,15,21, but stabilizes U6 small nuclear RNA18,19. The lengths of U tails required for such effects can be astonishingly short: 1-3 U's inhibits miR-26 function6. Identification of the substrates of PUPs is key to understanding the importance of uridylation and may also link PUPs to known biological processes, much as Lin28-dependent TUT4 activity on pre-miRNA links PUP activity to development, tumorigenesis, and other disease states4,11,37-39. The experiments in Aim 2 will be the first global analyses of the RNA substrates of PUPs. They will likely reveal new classes of RNAs subjected to PUP control and provide insight into PUP biological roles. The field is still in its early days of understanding how PUPs work, on which RNAs they act, and their biological significance. Yet already, it is clear that PUPs have key roles in a wide variety of disease states. Our studies will provide a strong molecular and genetic foundation for understanding the roles of PUPs in humans.